Ahmad et al. BMC Res Notes (2016) 9:477 DOI 10.1186/s13104-016-2271-4 BMC Research Notes RESEARCH ARTICLE Open Access Genetic diversity of the Tibetan antelope (Pantholops hodgsonii) population of Ladakh, India, its relationship with other populations and conservation implications Khursheed Ahmad1, Ved P. Kumar2, Bheem Dutt Joshi2, Mohamed Raza1, Parag Nigam3, Anzara Anjum Khan1 and Surendra P. Goyal2* Abstract Background: The Tibetan antelope (Pantholops hodgsonii), or chiru, is an endangered antelope, distributed in China [Xinjiang, Xizang, Qinghai, Zhuolaihu Lake (Breeding habitat)], and India (Aksai Chin and Ladakh). There is a global demand for the species prized wool, which is used in weaving shahtoosh shawls. Over the years, the population of the Tibetan antelope has drastically declined from more than a million to a few thousand individuals, mainly due to poaching. Field studies undertaken in Ladakh, India also indicated winter migration of the population to Tibet. Migra- tion between winter and calving habitats is well established to be female-biased across the Qinghai Tibetan Plateau (QTP). For effective conservation planning, genetic characterization is considered the best way to understand the likely impact of threats for ensuring the long-term viability of the population. In this regard, genetic characteristics of all Chinese populations are well-studied using mitochondrial and microsatellite markers, but information is lacking for the Indian population. Therefore, using the control region marker, we document for the first time the genetic variation of the Indian population of the Tibetan antelope, the extent of migration and its relationships with other populations of China. Results: The partial fragment of control region (259 bp) marker was successfully amplified in 30 Tibetan antelope samples that were collected from the Chang Chenmo Valley in eastern Ladakh, India. We also retrieved control region sequences (n 88) available in the public domain from GenBank of different Chinese populations. Low levels of nucleotide (π; =0.004) and haplotype (hd; 0.543) diversity were observed in the Indian population when compared to Chinese populations (π 0.01357–0.02048 and hd 0.889–0.986). Commonly used indices (Tajima’s D and Fu’s Fs) were analyzed for inferring= the demographic history= of the Indian populations, and all values were negative indicating population expansion or demographic equilibrium, though nonsignificant. We observed five haplotypes in the Indian population, and these were not reported in previously studied populations of QTP. Bayesian-based phylogenetic analysis indicates the presence of four clades, however, the posterior probability support for three of these clades is weak (<0.5). Of these, the Indian population formed a distinct clade, whereas the Chinese populations exhibited shared haplotypes, and no geographic structure was observed. Median-joining network analysis was conducted for 46 haplotypes in the overall population, except the samples from India which showed a star-like topology. The Indian population is separated by one median vector from the Chinese population. Conclusions: The present study revealed the presence of different sub-clades in the Bayesian phylogenetic tree and five new haplotypes only in the Indian population or sampling location. Furthermore, in the phylogenetic tree, *Correspondence: [email protected] 2 Wildlife Forensic and Conservation Genetics Cell, Wildlife Institute of India, Post Box #18, Chandrabani, Dehradun, Uttarakhand 248001, India Full list of author information is available at the end of the article © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ahmad et al. BMC Res Notes (2016) 9:477 Page 2 of 10 Indian haplotypes of Tibetan antelopes were clustered with the haplotype reported in the Chinese population of the Xinjiang region. Median-joining network analysis showed shared haplotypes pattern in all populations of QTP except the samples from India which showed new haplotypes. Given the presence of low nucleotide and haplotype diversity in eastern Ladakh populations and limited information available for populations of the western side in its range, we suggest to include genetic studies of Tibetan antelope populations around Aksai Chin (Fig. 1) under the proposed transboundary agenda between India and China and assess relationships with other populations. Such understand- ing would enable the planning of conservation strategies for ensuring long-term survival of westernmost populations in its range, and if required, it would establish connectivity with the other populations. Keywords: mtDNA, Shahtoosh wool, Poaching and conservation, Tibetan antelope Background The Tibetan antelope is endemic to the Qinghai The Tibetan antelope (Pantholops hodgsonii), also known Tibetan Plateau (QTP) and occupies open-high eleva- as chiru has very recently been reclassified on the Inter- tion alpine and desert steppe habitats in the mountainous national Union for Conservation of Nature (IUCN) Red terrain with frequent occurrence at elevations between list as near threatened due to the recovery of some popu- 3250 and 5500 m [3–6]. The present population occurs, lations [1]. The species is listed in Appendix 1 of the Con- almost exclusively, in about 800,000 km2 of the Chinese vention On International Trade In Endangered Species provinces of Xizang (XZ), Xinjiang (XJ), Qinghai (QH) (CITES) (1979) [2], and in Schedule I of the Indian Wild- [7–11], Zhuolaihu Lake (breeding habitat; BH), and in life (Protection) Act 1972. It is a Class I protected species very small numbers in north-western India (Aksai Chin under the Law of the People’s Republic of China on the and Ladakh), mainly in summer [12–14] (Fig. 1). The Protection of Wildlife (1989). Tibetan antelope has rarely been found in Nepal [15–18]. Fig. 1 Current distribution of Tibetan antelope (Pantholops hodgsonii) in the Tibetan Plateau of the Chinese provinces of Tibet, Qinghai, Xinjiang and India Ahmad et al. BMC Res Notes (2016) 9:477 Page 3 of 10 At the beginning of the last century, more than a mil- scarves [29–31]. These garments can be sold for $1000– lion Tibetan antelopes were present, but more than 50 % 5000 USD for a single piece and sometimes as much as of individuals have been reduced in just 20 years of the $15,000–17,600 USD depending on the length [31, 32]. 20th century due to habitat fragmentation and increased It has been documented that when large numbers of ani- human activity such as poaching, hunting and smuggling mals are harvested or removed from the wild population of product [5, 10, 11]. Approximately 75,000 Tibetan due to poaching and other factors, the genetic diversity antelopes are now reported in their distributional range and population structure is severely affected by decreas- [19, 20]. Zhuolaihu Lake is known as one of the major ing effective population size [33, 34]. breeding habitats (BH) or calving ground for Tibetan A limited number of studies have been conducted so far antelopes present in the Qinghai province where dur- on the genetic aspects of Tibetan antelope populations, ing the summer season, thousands of females migrate and these studies have revealed that Tibetan antelopes there from wintering habitats to breed [21]. It has been have female-biased dispersal with a high mutation rate of suggested that the migratory behavior exhibited by the mitochondrial DNA (mtDNA) [10, 19], which is a good Tibetan antelope promotes genetic exchange between marker for identifying female-based linkages between different meta-populations and maintains all the popula- populations [35–38]. Hence, we hypothesized that the tions together as one panmictic population [21]. Tibetan antelope population of India is also genetically In India, Tibetan antelopes are found in Daulat Beg connected with the rest of the populations because of Oldi (DBO) and the Chang Chenmo Valley (CCV) in the observed migratory behavior on QTP. Therefore, north–east Ladakh (Fig. 1) [13, 14]. The average altitude we describe, for the first time, genetic variation in the of these areas is 4725–5500 m, and Tibetan antelopes hypervariable control region of the mtDNA genome and are found between the altitudes of 4709–4964 m (mean describe its relationships with other populations. 4797 ± 27.9 m). These areas are mostly surrounded by glaciers and rivers. Unequal individual numbers were Methods sighted from the DBO: 230 individuals during 2005 and Sample collection 45 individuals in 2006. In the CCV, a total of 55–60 indi- Four hair samples and 26 fecal samples were collected viduals of Tibetan antelopes were reported during 2005. from different locations in Chang Chenmo Valley These two Indian populations (DBO and CCV) are iso- (Ladakh District), Jammu and Kashmir, India (IND 1–30) lated from one another due to mountains and river barri- (Table 1) during field work. Collected hair samples were ers. In DBO, both males and females have been reported kept in an envelope and supplied with the sampling loca- [22, 23], whereas in CCV, only males have been seen [11]. tion and sample ID. The fecal pellets were collected and Though migration from wintering to calving habitats is air dried to remove all moisture. The pellets were then well-documented on QTP, little is known for such move- kept in sterile vials containing silica gel and brought into ments of the population of India. It has been reported the laboratory. that the male Tibetan antelope population in CCV in India is resident [23]. In contrast to these males, individ- DNA extraction and PCR amplification ual females travel distances of 300–400 km in late spring Genomic DNA was extracted from hair samples using a and early summer [24].